The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P₂), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P₂ within μ2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant μ2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P₂ and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P₂-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant μ2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P₂ binding to μ2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.